Enhanced plant cell transformation by addition of host genes involved in T-DNA integration

ABSTRACT

Adding at least one gene involved in plant host cell T-DNA integration enhances transformation by  Agrobacterium . The histone H2A gene encoded by the  Arabidopsis  RAT5 gene increases transformation frequencies of plants, most likely by causing overexpression of a product needed for T-DNA integration.  Agrobacterium tumefaciens  genetically transforms plant cells by transferring a portion of the bacterial Ti-plasmid, designated the T-DNA, to the plant, and integrating the T-DNA into the plant genome. However, not all plants are transformable by  Agrobacterium  and transformation frequencies may be too low to be useful. Little is known about the T-DNA integration process, and no plant genes involved in integration have been identified prior to the present invention.

This application claims priority to U.S. provisional application Ser.No. 60/154,158 filed Sep. 15, 1999.

The U.S. government may have rights in this invention due to partialsupport from the National Science Foundation (IBN-9630779).

BACKGROUND

The invention relates enhanced Agrobacterium transformation frequenciesof plants due to overexpression of the histone H2A gene encoded by theArabidopsis RAT5 gene. Agrobacterium tumefaciens is a gram negative soilbacterium that has been exploited by plant biologists to introduceforeign DNA into plants. However, there are some limitations on the useof this transforming vector, e.g. difficulties in transforming monocots,and transforming frequencies may be too low to be useful. Although knownfor this practical application, the actual mechanism of DNA transferfrom bacteria to plants is not completely understood.

Agrobacterium tumefaciens genetically transforms plant cells bytransferring a portion of the bacterial Ti-plasmid, designated theT-DNA, to the plant, and integrating the T-DNA into the plant genome.Little is known about the T-DNA integration process, and no plant genesinvolved in integration have previously been identified. The DNA that istransferred from Agrobacterium to the plant cell is a segment of the Ti,or tumor inducing, plasmid called the T-DNA (transferred DNA). Virulence(vir) genes responsible for T-DNA processing and transfer are reportedto lie elsewhere on the Ti plasmid. The role of vir genes in T-DNAprocessing, the formation of bacterial channels for export of T-DNA, andthe attachment of bacteria to the plant cell are reported (Sheng andCitovsky, 1996; Zupan and Zambryski, 1997). In contrast, little is knownabout the role of plant factors in T-DNA transfer and integration. Theisolation of a putative plant factor has recently been reported. Ballasand Citovsky showed that a plant karyopherin α (AtKAP α) can interactwith VirD2 nuclear localization sequences in a yeast two-hybridinteraction system, and is presumably involved in nuclear translocationof the T-complex. Using a similar approach, a tomato type 2C proteinphosphatase, DIG3, that can interact with the VirD2 NLS was identified.Unlike AtKAP α, DIG3 plays a negative role in nuclear import. After theT-DNA/T-complex enters the nucleus, it must integrate into the plantchromosome. Plant chromosomal DNA is packaged into nucleosomesconsisting primarily of histone proteins. The incoming T-DNA may have tointeract with this nucleosome structure during the integration process.However, T-DNA may preferentially integrate into transcribed regions ofthe genome. These regions are believed to be temporarily free ofhistones. How exactly T-DNA integration takes place is unknown. Recentreports have implicated involvement of VirD2 protein in the T-DNAintegration process. Plant proteins are also likely to be involved inthis process (Deng et al., 1998; Ballas and Citovsky, 1997; Tao, etal.). Other evidence for the involvement of plant factors in T-DNAtransfer and integration comes from identification of several ecotypesof Arabidopsis that are resistant to Agrobacterium transformation.

To identify plant genes involved in Agrobacterium -mediatedtransformation, a T-DNA tagged Arabidopsis library was screened formutants that are resistant to Agrobacterium transformation (ratmutants). There are several steps in which plant genes are likelyinvolved in the Agrobacterium-mediated transformation process. First,plant-encoded factors could be involved in the initial step of bacterialattachment to the plant cell surface. Mutants and ecotypes that aredeficient in bacterial attachment have been identified and genesinvolved in bacterial attachment are currently being characterized. Thenext step in which a plant factor(s) could be involved is the transferof T-strands from the bacteria to plant cells across the plant cell walland membrane. After T-DNA/T-complex enters the cytoplasm of the plantcell, plant factors are required to transport the T-complex to thenucleus.

An Arabidopsis T-DNA tagged mutant, rat5, was characterized that isdeficient in T-DNA integration and is resistant toAgrobacterium-mediated root transformation. Both genetic and DNA blotanalyses indicated that there are two copies of T-DNA integrated as atandem repeat at a single locus in rat5. No major rearrangements are inthe rat5 plant DNA immediately surrounding the T-DNA insertion site.These data strongly suggest that in rat5 the T-DNA had inserted into agene necessary for Agrobacterium-mediated transformation. The sequenceof the T-DNA left border-plant junction indicated that the T-DNA hadinserted into the 3′ untranslated region of a histone H2A gene. Thisinsertion is upstream of the consensus polyadenylation signal. Byscreening an Arabidopsis ecotype Ws cDNA library and sequencing 20different histone H2A cDNA clones, and by performing a computer database search, at least six different histone H2A genes were shown. Thesegenes encode proteins that are greater than 90% identical at the aminoacid sequence level. Thus, the histone H2A genes comprise a smallmulti-gene family in Arabidopsis.

T-DNA integration does not appear to take place by homologousrecombination, believed to be the most common method of foreign DNAintegration in prokaryotes and lower eukaryotes, because no extensivehomology between the T-DNA and target sequences has been found. T-DNA isreported to integrate by illegitimate recombination (Matsumoto et al.,1990; Gheysen et al., 1991; Mayerhofer et al., 1991; Ohba et al., 1995).Illegitimate recombination is the predominant mechanism of DNAintegration into the genomes of higher plants (Britt, 1996; Offringa etal., 1990; Paszkowski et al., 1988).

Information on factors affecting Agrobacterium transformationfrequencies in plants is needed to improve performance of this method.

SUMMARY OF THE INVENTION

The invention relates to increased Agrobacterium transformationfrequencies in plants due to addition of at least one gene involved inhost T-DNA integration, to the host plant. In an embodiment, addition ofat least one histone H2A gene encoded by the Arabidopsis RAT5 geneenhances transformation frequencies, most likely due to overexpressingof histone as compound to the host's natural expression levels. The genecan be either in transgenic plants or carried by the transforming agent,T-DNA for practice of the invention.

Overexpression of histone genes of the present invention overcomes thepoor performance that limits the use of Agrobacterium as a transformingvector. Many plants can be transformed transiently by Agrobacterium sothey express the transforming gene for a period of time, but are notstably transformed because of T-DNA integration problems. Therefore,transgenic plants are not produced. The gene H2A (RAT5) plays animportant role in illegitimate recombination of T-DNA into the plantgenome and the gene's overexpression enhances transformation.

Transient and stable GUS (β-glucuronidase) expression data and theassessment of the amount of T-DNA integrated into the genomes ofwild-type and rat5 Arabidopsis plants indicated that the rat5 mutant isdeficient in T-DNA integration needed for transformation. Complementingthe rat5 mutation was accomplished by expressing the wild-type RAT5histone H2A gene in the mutant plant. Surprisingly, overexpression ofRAT5 in wild-type plants increased Agrobacterium transformationefficiency. Furthermore, transient expression of a RAT5 gene from theincoming T-DNA was sufficient to complement the rat5 mutant and toincrease the transformation efficiency of wild-type Arabidopsis plants.The present invention provides methods and compositions to increasestable transformation frequency in plants using direct involvement of aplant histone gene in T-DNA integration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows characteristics of the rat5 mutant: (A) stabletransformation of wild-type Arabidopsis ecotype Ws, the rat5 mutant, andthe F1 progeny; (B) sequence of the rat5/T-DNA junction region; (C)pattern of T-DNA integration in rat5: LB, T-DNA left border; RB, T-DNAright border; pBR322, pBR322 sequences containing the β-lactamase geneand ColE1 origin of replication; Tn903, kanamycin resistance gene for E.coli selection; Tn5, kanamycin resistance gene for plant selection.

FIG. 2 shows complementation of the rat5 mutant and overexpression ofRAT5 in wild-type Arabidopsis plants; maps of the binary vectors pKM4(A) and pKM5 (B) RB, T-DNA right border; LB, T-DNA left border; pAnos,nopaline synthase polyadenylation signal sequence; histone H2A, codingsequence of the RAT5 histone H2A gene; pH2A, promoter sequence of theRAT5 histone H2A gene; Pnos, nopaline synthase promoter; hpt, hygromycinresistance gene; pAg7, agropine synthase polyadenylation signalsequence; uidA, promoterless gusA gene; arrows above the histone H2A,uidA, and hpt genes indicate the direction of transcription; (C)complementation of the rat5 mutant; (D) tumorigenesis assay of Wstransgenic plants overexpressing the RAT5 histone H2A gene.

FIG. 3 shows T-DNA integration assays of rat5 and Ws plants; (A)transient and stable GUS expression in Ws and rat5; (B) T-DNAintegration in rat5 and Ws plants.

DESCRIPTION OF THE INVENTION

Several T-DNA tagged [plants which genes have randomly been disrupted byintegration of a T-DNA] mutants of Arabidopsis were identified that arerecalcitrant to Agrobacterium root transformation. These are called ratmutants (resistant to Agrobacterium transformation). In most of thesemutants Agrobacterium transformation is blocked at an early step, eitherduring bacterial attachment to the plant cell or prior to T-DNA nuclearimport. In some of the mutants, however, the T-DNA integration step ismost likely blocked. Because plant factors involved in illegitimaterecombination of T-DNA into the plant genome have not previously beenidentified, the characterization of a T-DNA tagged Arabidopsis mutant,rat5, that is deficient in T-DNA integration, is an aspect of thepresent invention.

Characterization of the rat5 mutant. rat5, an Arabidopsis T-DNA taggedmutant, was previously identified as resistant to Agrobacterium roottransformation. An in vitro root inoculation assay was performed usingthe wild-type Agrobacterium strain A208 (At10). After one month, thepercentage of root bundles that formed tumors was calculated. Greaterthan 90% of the root bundles of the wild-type plants (ecotype Ws) formedlarge green teratomas. In contrast, fewer than 10% of the root bundlesfrom the rat5 plants responded to infection, forming small yellow calli(FIG. 1A). A homozygous rat5 plant (pollen donor) was crossed to awild-type plant (egg donor) and the resulting F1 progeny tested forsusceptibility to Agrobacterium transformation. This analysis indicatedthat rat5 is a dominant mutation (7; FIG. 1A). Further analysis of F2progeny indicated that kanamycin resistance segregated 3:1, indicatingthat a single locus had been disrupted by the mutagenizing T-DNA.Kanamycin resistance co-segregated with the rat5 phenotype, indicatingthat a gene involved in Agrobacterium transformation had most likelybeen mutated by the T-DNA insertion.

Recovery of a T-DNA-plant junction from rat5. The T-DNA integrationpattern in the rat5 mutant was determined by DNA blot analyses. Theresults indicated that there are only two copies of the mutagenizingT-DNA integrated into the genome of the rat5 mutant. Further analysisindicated that these two T-DNA copies are present as a direct tandemrepeat, as shown in FIG. 1C.

A left border (LB) T-DNA-plant junction was recovered from rat5 using aplasmid rescue technique (see Materials and Methods) and a restrictionendonuclease map of this T-DNA-plant junction was constructed. Anapproximately 1.7 kbp EcoRI fragment that contains both plant and LB DNAwas subcloned into pBluescript and subsequently sequenced at the PurdueUniversity sequencing center. The sequence of this fragment is shown inFIG. 1B. DNA sequence analysis of this junction region indicated thatthe T-DNA had inserted into the 3′ untranslated region (UTR) of ahistone H2A gene (FIG. 1B). The histone H2A genes of Arabidopsis werefurther characterized by isolating and sequencing numerous cDNA andgenomic clones. Six different gene variants of histone H2A wereidentified, indicating that the histone H2A genes of Arabidopsiscomprise a small multi-gene family. In a lambda genomic DNA library aclone was identified containing the wild-type histone H2A genecorresponding to RAT5. DNA sequence analysis of this genomic cloneindicated that in rat5 the T-DNA had inserted upstream of the consensuspolyadenylation signal (AATAA). DNA blot analysis of Ws and rat5 DNAindicated that the T-DNA insertion in rat5 did not cause any majorrearrangements in the plant DNA immediately around the site ofinsertion. Disruption of the 3′ UTR of the RAT5 histone H2A gene islikely the sole cause for the rat phenotype in the rat5 mutant.

FIG. 1 shows characterization of the rat5 mutant. (A) Stabletransformation of wild-type Arabidopsis ecotype Ws, the rat5 mutant, andthe F1 progeny. Sterile root segments were infected with A. tumefaciensA208. Two days after cocultivation, the roots were transferred to MSmedium lacking phytohormones and containing timentin as an antibiotic.Tumors were scored after four weeks. (B) Sequence of the rat5/T-DNAjunction region. (C) Pattern of T-DNA integration in rat5. LB, T-DNAleft border; RB, T-DNA right border; pBR322, pBR322 sequences containingthe β-lactamase gene and ColE1 origin of replication; Tn903, kanamycinresistance gene for E. coli selection; Tn5, kanamycin resistance genefor plant selection. Five μg of genomic DNA from the rat5 mutant wasdigested with either EcoRI or SalI and was blotted onto a nylonmembrane. An EcoRI-SalI fragment of pBR322 was used as the hybridizationprobe. Restriction fragment sizes shown above the T-DNA were detected byEcoRI digestion and the sizes shown below the T-DNA were detected bySalI digestion.

Complementation of the rat5 mutant with a wild-type histone H2A gene(RAT5). Two different constructions were made to perform acomplementation analysis of the rat5 mutant. First, a nopaline synthaseterminator (3′ NOS) was fused to the 3′ region of the 1.7 kbp junctionfragment (the sequence of this 1.7 kbp fragment is shown in FIG. 1B).This construction contains the RAT5 histone H2A gene with its ownpromoter and a 3′ NOS. This fragment (RAT5 plus 3′ NOS) was cloned intothe binary vector pGTV-HPT of beaker containing a hygromycin resistancegene between the left and the right T-DNA borders, resulting in thebinary vector pKM⁴ (FIG. 2A). For the second construction, a 9.0 kbpSacI genomic fragment of wild-type Ws DNA containing a histone H2A gene(RAT5) plus at least 2.0 kbp sequences upstream and downstream of RAT5was cloned into the binary vector pGTV-HPT, resulting in the binaryvector pKM5 (FIG. 2B). pKM4 and pKM5 were transferred separately intothe non-tumorigenic Agrobacterium strain GV3101, resulting in strains A.tumefaciens At1012 and At1062, respectively.

Both strains At1012 and At1062 were separately used to transform rat5plants using a germ-line transformation method (Bent et al., 1998) andtransgenic rat5 plants were selected for resistance to hygromicin (20μg/ml). Several transgenic plants (T1) were obtained. These transgenicplants were allowed to self fertilize and T1 seeds were collected. Sixtransgenic lines obtained by transformation with At1012 (the wild-typehistone H2A with 3′ NOS) were randomly selected and their seeds weregerminated in the presence of hygromycin. Tumorigenesis assays wereperformed as described in Nam et al. (1999) using A. tumefaciens At10and a sterile root inoculation protocol, on at least five differentplants from each of the six transgenic lines. The results indicated thatin five of the six transgenic rat5 lines tested, thetumorigenesis-susceptibility phenotype was recovered (FIG. 2C; Table 1).Teratomas incited on the roots of these plants appeared similar totumors generated on a wild-type plant. One of the transgenic plantstested did not recover the tumorigenesis-susceptibility phenotype,probably because of an inactive transgene. Transgenic T1 plants of rat5obtained by transformation with At1062 (containing a genomic encodingRAT5 from the wild-type plant) were also tested for restoration of thetumorigenesis-susceptibility phenotype. Some of these plants were alsoable to recover the tumorigenesis-susceptibility phenotype, indicatingcomplementation of the rat5 mutation. Hygromycin-resistant transgenicplants generated by transforming the rat5 mutant with pGPTV-HPT alonedid not form tumors upon infection with A. tumefaciens A208.

To confirm the genetic basis of the complementation experiment, aco-segregation analysis was performed on one of the rat5 transgeniclines (rat5 At1012-6) obtained by transformation of the rat5 mutant withA. tumefaciens At1012. To examine the co-segregation of thecomplementing T-DNA containing the wild-type RAT5 gene with thetumorigenesis-susceptibility phenotype, seeds from a T2 plant homozygousfor the rat5 mutation but heterozygous for hygromycin resistance weregerminated and grown on B5 medium without selection. Roots of theseplants were subsequently tested for hygromycin-resistance andsusceptibility to crown gall tumorigenesis. All plants that weresensitive to hygromycin were also resistant to tumor formation in amanner similar to that of the rat5 mutant. Of the 25hygromycin-resistant plants, at least 8 were susceptible totumorigenesis. However, 17 hygromycin-resistant plants remainedrecalcitrant to Agrobacterium-mediated transformation. It is likely thatthese plants are heterozygous with respect to the complementing RAT5gene and did not express this gene to a level high enough to restoresusceptibility to tumorigenesis. This possibility corresponds to thefinding that the rat5 mutation is dominant, and that therefore oneactive copy of RAT5 is not sufficient to permit Agrobacterium-mediatedtransformation. Taken together, the molecular and genetic data stronglyindicate that in the rat5 mutant disruption of a histone H2A gene isresponsible for the tumorigenesis-deficiency (rat) phenotype.

Overexpression of a histone H2A (RAT5) gene in wild-type plants improvesthe efficiency of Agrobacterium transformation. To determine furtherwhether the RAT5 gene plays a direct role in Agrobacterium-mediatedtransformation,A. tumefaciens At1012 was used to generate severaltransgenic Arabidopsis plants (ecotype Ws) containing additional copiesof the RAT5 histone H2A gene. These transgenic plants were allowed toself-pollinate, T1 seeds were collected, and T2 plants were germinatedin the presence of hygromycin. Tumorigenesis assays were performed asdescribed herein at least five plants from each of four differenttransgenic lines. Because ecotype Ws normally is highly susceptible toAgrobacterium transformation, the tumorigenesis assay was altered todetect any subtle differences between the transformation-susceptiblewild-type plant and transgenic wild-type plants overexpressing RAT5.These alterations included inoculation of root segments with a 100-foldlower concentration (2×10⁷ cfu/ml) of bacteria than that normally used(2×10⁹ cfu/ml), and spreading individual root segments rather thanbundles of root segments on MS medium to observe tumor production. Theresults, shown in Table 1 and FIG. 2D, indicate that transgenic plantsoverexpressing RAT5 are approximately twice as susceptible to roottransformation as are wild-type Ws plants. These data indicate that theRAT5 histone H2A gene plays a direct role in T-DNA transformation, andthat overexpression of RAT5 can increase susceptibility totransformation.

Transient expression of histone H2A is sufficient to permittransformation of rat5 and to increase the transformation efficiency ofwild-type Ws plants. Expression of the RAT5 histone H2A gene from theincoming T-DNA complement the rat5 mutant. Although transformation ofthis mutant with an Agrobacterium strain harboring pGPTV-HYG (lacking ahistone H2A gene) resulted in only a few, slow-growing calli onhygromycin selection medium, Agrobacterium strains harboring pKM4 orpKM5 incited rapidly growing hygromycin-resistant calli on 60±21% and54±22% of the rat5 root segment bundles, respectively. In addition, whenwild-type plants were infected (at low bacterial density) with atumorigenic Agrobacterium strain (A208) harboring pKM4, 78±8% of theroot segments developed tumors, compared to 36±9% of the root segmentsinfected with a tumorigenic bacterial strain harboring pGPTV-HYG. Thesetransformation experiments indicate that Agrobacterium strainscontaining the binary vectors pKM4 or pKM5 are able to transform rat5mutant plants at relatively high efficiency, and on wild-type plants aretwo-fold more tumorigenic, and are better able to incitehygromycin-resistant calli, than are Agrobacterium strains containingthe “empty” binary vector pGPTV-HYG. Transiently produced histone H2Amay improve the stable transformation efficiency of plants byAgrobacterium.

The rat5 mutant is deficient in T-DNA integration.Agrobacterium-mediated transformation of the Arabidopsis rat5 mutantresults in a high efficiency of transient transformation but a lowefficiency of stable transformation, as determined by the expression ofa gusA gene encoded by the T-DNA. This result suggested that rat5 ismost likely deficient in T-DNA integration. To test this hypothesisdirectly root segments from Ws and rat5 plants were inoculated with A.tumefaciens GV3101 harboring the T-DNA binary vector pBISN1. pBISN1contains a gusA-intron gene under the control of a “super-promoter” (Niet al., 1995; Narasimhulu et al., 1996). Two days after cocultivation,the root segments were transferred to callus inducing medium containingtimentin (100 μg/ml) to kill the bacteria. Three days after infection, afew segments were stained for GUS activity using the chromogenic dyeX-gluc. Both the wild-type and the rat5 mutant showed high levels of GUSexpression (approximately 90% of the root segments stained blue; FIG.3A). The remaining root segments were allowed to form calli on callusinducing medium containing timentin to kill Agrobacterium, but lackingany antibiotic for selection of plant transformation. After four weeksnumerous calli derived from at least five different Ws and rat5 plantswere stained with X-gluc. Of the Ws calli sampled, 92±12% showed largeblue staining areas, whereas only 26±10% of the rat5 calli showed GUSactivity, and most of these blue staining regions were small (FIG. 3A).These data indicate that although the rat5 mutant can transientlyexpress the gusA gene at high levels, it fails to stabilize gusAexpression.

Suspension cell lines were generated from these Ws and rat5 calli andafter an additional month the amount of T-DNA was assayed (using as ahybridization probe the gusA-intron gene located within the T-DNA ofpBISN1) integrated into high molecular weight plant DNA from Ws and rat5calli (Nam et al., 1997; Mysore et al., 1998). FIG. 3B shows thatalthough T-DNA integrated into the genome of wild-type Ws plants waseasily detectable, T-DNA integrated into the rat5 genome was not. Thesedata directly demonstrate that rat5 is deficient in T-DNA integration.To demonstrate equal loading of plant DNA in each of the lanes, the gusAprobe was stripped from the blot and rehybridized the blot with anArabidopsis phenylalanine ammonia-lyase (PAL) gene probe.

FIG. 2 shows complementation of the rat5 mutant and overexpression ofRAT5 in wild-type Arabidopsis plants. Maps of the binary vectors pKM4(A) and pKM5 (B). RB, T-DNA right border; LB, T-DNA left border; pAnos,nopaline synthase polyadenylation signal sequence; histone H2A, codingsequence of the RAT5 histone H2A gene; pH2A, promoter sequence of theRAT5 histone H2A gene; Pnos, nopaline synthase promoter; hpt, hygromycinresistance gene; pAg7, agropine synthase polyadenylation signalsequence; uidA, promoterless gusA gene. Arrows above the histone H2A,uidA, and hpt genes indicate the direction of transcription. (C)Complementation of the rat5 mutant. rat5 mutant plants were transformedwith an Agrobacterium strain containing the binary vector pKM4 (At1012).Hygromycin-resistant transgenic plants were obtained and wereself-pollinated to obtain T2 plants. Sterile root segments of T2 plantsexpressing RAT5, wild-type Ws plants, and rat5 mutant plants wereinfected with the tumorigenic strain A. tumefaciens A208. Two days aftercocultivation, the roots were moved to MS medium lacking phytohormonesand containing timentin. Tumors were scored after four weeks. (D)Tumorigenesis assay of Ws transgenic plants overexpressing the RAT5histone H2A gene. Ws plants were transformed with A. tumefaciens At1012containing the binary vector pKM4. Hygromycin-resistant transgenicplants were obtained and were self-pollinated to obtain T2 plants.Sterile root segments of T2 plants overexpressing RAT5 and wild-type Wsplants were infected at low bacterial density with A. tumefaciens A208.After two days cocultivation, the roots were moved to MS medium lackingphytohormones and containing timentin. Tumors were scored after fourweeks.

Teratomas incited on the roots of these plants appeared similar totumors generated on a wild-type plant. One of the transgenic plantstested did not recover the tumorigenesis-susceptibility phenotype,probably because of an inactive transgene. Transgenic T1 plants of rat5obtained by transformation with At1062 (containing a genomic encodingRAT5 from the wild-type plant) were also tested for restoration of thetumorigenesis-susceptibility phenotype. Some of these plants were alsoable to recover the tumorigenesis-susceptibility phenotype, indicatingcomplementation of the rat5 mutation. Hygromycin-resistant transgenicplants generated by transforming the rat5 mutant with pGPTV-HPT alonedid not form tumors upon infection with A. tumefaciens A208.

FIG. 3 shows T-DNA integration assays of rat5 and Ws plants; (A)transient and stable GUS expression in Ws and rat5; Sterile rootsegments of Ws and rat5 plants were infected with the non-tumorigenicAgrobacterium strain GV3101 containing the binary vector pBISN1. Twodays after cocultivation, the roots were transferred to callus inducingmedium (CIM) containing timentin. Three days after infection, half ofthe segments were stained with X-gluc to determine the efficiency oftransient GUS expression. The other group of segments was allowed toform calli on CIM. After four weeks these calli were stained with X-glucto determine the efficiency of stable GUS expression. (B) T-DNAintegration in rat5 and Ws plants. Suspension cells were derived fromthe calli generated from Ws and rat5 root segments infected with thenon-tumorigenic Agrobacterium strain GV3101 containing the binary vectorpBISN1. The suspension cell lines were grown for three weeks (withoutselection for transformation) in the presence of timentin or cefotaximeto kill Agrobacterium. Genomic DNA was isolated from these cells,subjected to electrophoresis through a 0.6% agarose gel, blotted onto anylon membrane, and hybridized with a gusA gene probe. Afterautoradiography, the membrane was stripped and rehybridized with aphenylalanine ammonia-lyase (PAL) gene probe to determine equal loadingof DNA in each lane.

Materials and Methods

Nucleic acid manipulation. Total plant genomic DNA was isolatedaccording to the method of Dellaporta et al. (1983). Restrictionendonuclease digestions, agarose gel electrophoresis, plasmid isolation,and DNA blot analysis were conducted as described (Sambrook et al.,1982).

Plasmid Rescue. Genomic DNA (5 μg) of rat5 was digested to completionwith SalI. The digested DNA was extracted with phenol/chloroform andprecipitated with ethanol. The DNA was self-ligated in a final volume of500 μl in 1× ligation buffer (Promega) with 3 units of T4 DNA ligase at16° C. for 16 hr. The ligation mixture was precipitated with ethanol,transformed into electrocompetent E. coli DH10B cells (mcrBC-; LifeTechnologies, Inc., Gaithersburg, Md.) by electroporation (25 μF, 200 Ω,and 2.5 kV) and plated on LB medium containing ampicillin (100 μg/ml).Ampicillin-resistant colonies were lifted onto a nylon membrane, thebacteria were lysed, and DNA was denatured in situ (Sambrook et al.,1982). A radiolabeled left border (LB) sequence (3.0 kbp EcoRI fragmentof pE1461) was used as a hybridization probe to identify a plasmidcontaining the LB. Positive colonies were picked and plasmid DNA wasisolated. By restriction fragment analysis a plasmid containing both theLB and plant junction DNA was identified. The plant junction fragmentwas confirmed by hybridizing the junction fragment to wild-type plantDNA. A restriction map of this plasmid, containing the LB-plant junctionDNA, was made. A 1.7 kbp EcoRI fragment that contained plant DNA plus 75base pairs of LB sequence was subcloned into pBluescript, resulting-inpE1509. This fragment was subsequently sequenced at the PurdueUniversity sequencing center.

Growth of Agrobacterium and in vitro root inoculation of Arabidopsisthaliana These were performed as described previously by Nam et al.(1997).

Plant Growth Conditions Seeds of various Arabidopsis thaliana ecotypeswere obtained from S. Leisner and E. Ashworth (originally from theArabidopsis Stock Centre, Nottingham, UK, and the Arabidopsis BiologicalResource Center, Ohio State University, Columbus, respectively). Seedswere surface sterilized with a solution composed of 50% commercialbleach and 0.1% SDS for 10 min and then rinsed five times with steriledistilled water. The seeds were germinated in Petri dishes containingGamborg's B5 medium (GIBCO) solidified with 0.75% bactoagar (Difco). Theplates were incubated initially at 4° C. for 2 days and the fro 7 daysunder a 16-hr-lights/8-hr-dark photoperiod at 25° C. Seedlings wereindividually transferred into baby food jars containing solidified B5medium and grown for 7 to 10 days for root culture. Alternatively, theseedlings were transferred into soil for bolt inoculation.

Growth of Agrobacterium tumerfaciens All Agrobacterium strains weregrown in YEP medium (Lichtenstein and Draper, 1986) supplemented withthe appropriate antibiotics (rifampicin, 10 μg/mL; kanamycin, 100 μg/mL)at 30° C. Overnight bacterial cultures were washed with 0.9% NaCl andresuspended in 0.9% NaCl a 2×10⁹ colony-forming units per mL for invitro root inoculation or at 2×10¹¹ colony-forming units per mL for boltinoculation.

In Vitro Root Inoculation and Transformation Assays Roots grown on theagar surface were excised, cut into small segments (˜0.5 cm) in a smallamount of sterile water, and blotted onto sterile filter paper to removeexcess water. For some experiments, excised roots were preincubated oncallus-inducing medium (CIM;4.32 g/L Murashige and Skoog [MS] minimalsalts [GIBCO], 0.5 g/L Mes, pH 5.7, 1 mL/L vitamin stock solution [0.5mg/mL nicotinic acid, 0.5 mg/mL pyridoxine, and 0.5 mg/mL thyamine-HCl],100 mg/L myoinositol, 20 g/L glucose, 0.5 mg/L 2,4-dichlorophenoxyaceticacid, 0.3 mg/L kinetin, 5mg/L indoleacetic acid, and 0.75% bactoagar)for 1 day before cutting them into segments. Dried bundles of rootsegments were transferred to MS basal medium (4.32 g/L MS minimal salts,0.5 g/L Mes, pH 5.7, 1 mL/L vitamin stock solution, 100 mg/Lmyoinositol, 10 g/L sucrose and 0.75% bactoagar), and 2 or 3 drops ofbacterial suspension were placed on them. After 10 min, most of thebacterial solution was removed, and the bacteria and root segments werecocultivated at 25° C. for 2 days.

For transient transformation assays, the root bundles were infected withAgrobacterium strain GV3101 was used (Koncz and Schell, 1986) containingthe binary vector pBISN1 (Narasimhulu et al., 1996). After variousperiods of time, the roots were rinsed with water, blotted on filterpaper, and stained with X-gluc staining solution (50 mM NaH₂HPO₄, 10 mMNa₂ EDTA, 300 mM mannitol, and 2 mM X-gluc, pH 7.0) for 1 day at 37° C.For quantitative measurements of 0-glucuronidase (GUS) activity, theroots were ground in a microcentrifuge tube containing GUS extractionbuffer (50 mM Na₂HPO₄, 5 mM DTT, 1 mM Na₂ EDTA, 0.1% sarcosyl, and 0.1%Triton X-100, pH 7.0), and GUS specific activity was measured accordingto Jefferson et al. (1987).

To quantitate tumorigenesis, root bundles were infected with wild-typeAgrobacterium strains. After 2 days, the root bundles were rubbed on theagar surface to remove excess bacteria and then washed with sterilewater containing timentin (100 μg/mL). Individual root segments (initialassay) or small root bundles (5 to 10 root segments; modified assay)were transferred onto MS basal medium lacking hormones but containingtimentin (100 μg/mL) and incubated for 4 weeks.

For transformation of root segments to kanamycin resistance, rootbundles were inoculated with Agrobacterium strain GV3101 containingpBISN 1. After 2 days, small root bundles (or individual root segments)were transferred onto CIM containing timentin (100 μg/mL) and kanamycin(50 μg/mL). Kanamycin-resistant calli were scored after 4 weeks ofincubation at 25° C.

To determine stable GUS expression, roots were inoculated as given aboveand the root segments were transferred after 2 days to CIM containingtimentin (100 μg/mL) without any selection. After 4 weeks, GUS activitywas assayed either by staining with X-gluc or by measuring GUS specificactivity by using a 4-methylumbelliferyl β-D galactoside (MUG)fluorometric assay, as described above.

To determine the kinetics of GUS expression, root bundles were infected,the root segments were transferred after 2 days to CIM containingtimentin (100 μg/mL), and calli were grown on CIM without selection.Root bundles were assayed at various times, using a MUG fluorometricassay as described above, to measure GUS specific activity.

Construction of the binary vectors pKM4 and pKM5. The plasmid pE1509containing the 1.7 kbp junction fragment cloned into pBluescript wasdigested with EcoRI to release the junction fragment. The 5′ overhangingends were filled in using the Klenow fragment of DNA polymerase I anddeoxynucleotide triphosphates. The T-DNA binary vector (pE1011) pGTV-HPT(Becker et al., 1992) was digested with the enzymes SacI and SmaI,releasing the promoterless gusA gene from pGTV-HPT. The 3′ overhangingsequence of the larger fragment containing the origin of replication andthe hygromycin resistance gene (hpt) were removed using the 3′-5′exonuclease activity of Klenow DNA polymerase, and the resulting 1.7 kbpblunt end fragment was ligated to the blunt ends of the binary vector. Abinary vector plasmid containing the 1.7 kbp fragment in the correctorientation (pAnos downstream of the histone H2A gene) was selected andnamed pKM4 (strain E1547).

An approximately 9.0 kbp wild-type genomic SacI fragment containing thehistone H2A gene (RAT5) from a lambda genomic clone was cloned into theSacI site of the plasmid pBluescript. This 9.0 kbp SacI fragment wassubsequently released from pBluescript by digestion with SacI and wascloned into the SacI site of the binary vector pGTV-HPT, resulting inthe plasmid pKM5 (strain E1596). Both pKM4 and pKM5 were separatelytransferred by triparental mating (Ditta et al., 1980) into thenon-tumorigenic Agrobacterium strain GV3101, resulting in the strains A.tumefaciens At1012 and At1062, respectively.

Germ-line transformation of Arabidopsis. Germ-line transformations wereperformed as described in (Bent and Clough, 1998). Transgenic plantswere selected on B5 medium containing hygromycin (20 μg/ml).

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Zupan, J. R. & Zambryski, P. The Agrobacterium DNA transfer complex.Crit. Rev. Plant Sci. 16, 279-295 (1997). TABLE 1 Complementation of therat5 mutant and overexpression of RAT5 in wild-type (Ws) Arabidopsisplants % Root Bundles Line^(a) With Tumors Tumor morphology rat5complementation with At1012 (T2 plants)^(a) Ws 98 ± 2 large, green rat521 ± 6 small, yellow rat5 At1012-1 64 ± 30 large + small, green rat5At1012-2 17 ± 4 small, yellow rat5 At1012-3 70 ± 20 large + medium,green rat5 At1012-4 86 ± 6 large, green rat5 At1012-5 82 ± 10 large,green rat5 At1012-6 92 ± 5 large, green Overexpression of RAT5 in Ws (T2plants)^(ab) Ws 35 ± 14 large, green Ws At1012-1 69 ± 27 large, green WsAt1012-2 68 ± 25 large, green Ws At1012-3 64 ± 13 large, green WsAt1012-4 63 ± 20 large, green^(a)at least 5 plants were tested for each mutant and 40-50 root bundleswere tested for each plant^(b) Agrobacterium was diluted to a concentration 100-fold lower thanthat normally used, and single root segments were separated

1-3. (canceled)
 4. A transgenic plant comprising at least one additionalcopy of the RAT5 gene of Arabidopsis. 5-9. (canceled)
 10. A host celltransformed by at least one copy of a gene involved in T-cellintegration.
 11. The host cell of claim 10, wherein the gene is capableof overexpressing histone to enhance plant transformation.
 12. The hostcell of claim 11, wherein the gene is the RAT5 gene of Arabidopsis.